DNA polymerases are used in many biotechnological applications such as DNA sequencing and the polymerase chain reaction. DNA polymerases that have high accuracy or fidelity are desired for these applications. A simple method for measuring the fidelity of the DNA polymerases is therefore also desired.
One common approach to measure fidelity uses a polymerase to fill a gap in the bacteriophage M13mp2 lacZα gene fragment, which encodes the α-peptide, an inactive segment of β-galactosidase. When accurately copied, and subsequently introduced into E. coli that bears a complementing copy of the remaining β-galactosidase gene, functional β-galactosidase is reconstituted, resulting in the hydrolysis of X-gal and blue bacterial plaques (see Bebenek K., Kunkel T. A. Methods Enzymol. 1995; 262:217-232). Inaccurate polymerase activity may result in a defective α-peptide, eventually resulting in reduced or abolished β-galactosidase activity, indicated by light blue or colorless plaques. The error rate is calculated from the blue/colorless plaque ratio, and mutations can be determined by DNA sequencing. Although this method is widely used to assess DNA polymerase fidelity, the method is labor and time intensive.
A plasmid-based DNA polymerase assay has also been developed (see Keith B. J., Jozwiakowski S. K., and Connolly B. A. Anal. Biochem. 2013 Feb. 15; 433(2): 153-161). The assay is based on gapped plasmid containing the lacZα reporter gene in a single-stranded region. Nicking at two sites flanking lacZa, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. The accuracy of a polymerase can be determined by copying the gene in vitro and then introducing the plasmid into E. coli, an approach similar in concept to that described above for the bacteriophage system. The error rate is again calculated from the blue/colorlessplaque ratio. The plasmid system is an improvement over the bacteriophage system with respect to the gapped DNA template preparation step but the blue/colorless colony scoring step remains time-consuming and tedious.